Abstract

INTRODUCTION:

Deep burn wounds undergo a dynamic process known as wound progression that results in a deepening and extension of the initial burn area. The zone of stasis is more likely to develop more severe during wound progression in the presence of hypoperfusion. Hydrogen has been reported to alleviate injury triggered by ischaemia/reperfusion and burns in various organs by selectively quenching oxygen free radicals. The aim of this study was to investigate the possible protective effects of hydrogen against early burn-wound progression.

METHODS:

Deep-burn models were established through contact with a boiled, rectangular, brass comb for 20 s. Fifty-six Sprague-Dawley rats were randomly divided into sham, burn plus saline, and burn plus hydrogen-rich saline (HS) groups with sacrifice and analysis at various time windows (6 h, 24 h, 48 h) post burn. Indexes of oxidative stress, apoptosis and autophagy were measured in each group. The zone of stasis was evaluated using immunofluorescence staining, ELISA, and Western blot to explore the underlying effects and mechanisms post burn.

RESULTS:

The burn-induced increase in malondialdehyde was markedly reduced with HS, while the activities of endogenous antioxidant enzymes were significantly increased. Moreover, HS treatment attenuated increases in apoptosis and autophagy postburn in wounds, according to the TUNEL staining results and the expression analysis of Bax, Bcl-2, caspase-3, Beclin-1 and Atg-5 proteins. Additionally, HS lowered the level of myeloperoxidase and expression of TNF-α, IL-1β, and IL-6 in the zone of stasis while augmenting IL-10. The elevated levels of Akt phosphorylation and NF-κB p65 expression post burn were also downregulated by HS management.

CONCLUSION:

Hydrogen can attenuate early wound progression following deep burn injury. The beneficial effect of hydrogen was mediated by attenuating oxidative stress, which inhibited apoptosis and inflammation, and the Akt/NF-κB signalling pathway may be involved in regulating the release of inflammatory cytokines.

Representative clinical images and HE staining of burn models with saline and HS treatment.

The interspaces between two burn zones became narrower and were inclined to merge in the burn+saline groups over time, whereas the interspaces remained relatively stable at various time points with HS treatment (small and amplified square frames) (A). The dotted line indicated the progression boundary, and the regions labelled by asterisks presented the obvious hyalinization, which received direct thermal injury. Some changes in the zone of stasis (right part aside dotted line), such as disturbed constructions of the epidermis and inflammatory cell infiltration (small and amplified square frames), can be observed in the burn+saline groups, whereas HS treatment improved these changes at the late time points (24 h and 48 h) (B) (100× magnification). The sample size was n = 8 for each group.

Assessment of oxidative stress within the zone of stasis and the effects of HS.

The levels of MDA in the interspaces of various burn+saline groups presented remarkable augmentations after burn, which coincided with obvious declines in the activities of endogenous antioxidant enzymes (SOD, GSH-Px, CAT). Application of HS postburn led to the observation that significant reductions in the MDA levels and increases in the activities of antioxidant enzymes appeared at a range of time windows. The sample size was n = 6 for each group. The results were expressed as the mean±SEM.*p<0.05, **p<0.01, versus burn+saline; #p<0.05, ##p<0.01, versus sham. B+S, burn+saline; B+HS, burn+hydrogen-rich saline.

The representative immunohistochemistry staining and ELISA detection of MPO within the zone of stasis.

Compared with the sham group, apparent positive staining (brown stained tissues indicated by black arrows) was observed after burn insults at 6 h, 24 h, 48 h (upper row), while HS treatment seemed to decrease the number of positive cells (lower row)(400× magnification). The ELISA detection of MPO in the interspace skin tissues expressed similarly obvious elevations in the MPO level in various burn+saline groups, and a marked effect of HS treatment on decreasing these elevations were also found in the time-paired burn+HS groups (B). The sample size was n = 6 for each group. The results were expressed as the mean±SEM. **p<0.01, versus burn+saline; ##p<0.01, versus sham. B+S, burn+saline; B+HS, burn+hydrogen-rich saline.

Western blot analysis of inflammatory cytokines in wound interspaces after burn with HS treatment.

Parallel increases in TNF-α, IL-1β, and IL-6 protein expression (as proinflammatory cytokines) were found at the three time points via western blot, while IL-10 also increased after burn to mediate the anti-inflammatory response. HS administration reduced the elevations of the three selected pro-inflammatory cytokines, and it further raised the levels of IL-10 to enhance the anti-inflammatory effect in wound tissues. The sample size was n = 6 for each group. The results were expressed as the mean±SEM. *p<0.05, **p<0.01, versus burn+saline; #p<0.05, ##p<0.01, versus sham. B+S, burn+saline; B+HS, burn+hydrogen-rich saline.

Evaluation of apoptosis in the zone of stasis after burns with HS management.

Classic TUNEL staining indicated symbolic brown nuclei that were mainly located in the dermis of the interspace skin postburn (400× magnification)(A). Counting the apoptotic cells indicated a significant elevation in the burn controls over time, with a peak occurring at 72 h post burn. Except for the 6 h group, HS administration effectively decreased the number of apoptotic cells in the treatment groups at 24 h and 48 h (400× magnification)(A). Bcl-2 expression was downregulated after burn insult, while the protein expression of Bax and caspase-3 exhibited increasing trends over time (B-E). Nevertheless, HS seemed to reverse these of expression tendencies at various time points (B-E). The sample size was n = 5 for each group. The results were expressed as the mean±SEM. *p<0.05, **p<0.01, versus burn+saline; #p<0.05, ##p<0.01, versus sham. B+S, burn+saline; B+HS, burn+hydrogen-rich saline.

The phosphorylation of Akt in the zone of stasis increased markedly at the three time windows after burn, and the peak occurred at 24 h. Similarly, the expression of NF-κB p65 underwent a significant elevation post burn. HS application downregulated both the elevation in phosphorylated Akt and that of NF-κB p65 expression. The sample size was n = 6 for each group. The results were expressed as the mean±SEM. **p<0.01, versus burn+saline; ##p<0.01, versus sham. B+S, burn+saline; B+HS, burn+hydrogen-rich saline.